Fish lose their way with nostrils on acid

Scientists examine the effect of increasing ocean acidity on the ability of …

Baby clownfish spend their first 11-12 days of their life in deep water, but need to find a reef to live out their adult life. It is thought that they find the reef by following their noses home, but what happens when you add a little acid to the water?

Like it or not, we are adding acid to the world's oceans�via dissolved CO2, a�byproduct of all the�CO2�we've been putting into the atmosphere. This acid is believed to be causing problems for coral reefs, and a new study in the Proceedings of the National Academy of Science looks at its effect on a fish's sense of smell, in part by performing electron microscopy of fish nostrils.

The researchers raised hundreds of baby clownfish for 11 days, then split them into two test groups and one control group. The test groups were raised in water with a pH of 7.8 and 7.6, while the control group was raised in water with a pH of 8.15. 8.15 is the pH of modern ocean water, while 7.8 is the pH we can expect for ocean water around the year 2100 under a business-as-usual scenario; 7.6 is the expected pH in 2200. They created the test conditions by bubbling CO2 through standard ocean water.

To test the fish's olfactory capabilities, they let them swim in a flume with two streams of water coming in, one with a favorable smell and the other with a neutral or unfavorable smell. The favorable smell was a heady mix of sea anemone, beneficial tree leaves, and eau de clownfish. The neutral smells were unscented sea water and a grass that is expected to have no benefit (and perhaps some drawbacks) for the fish. The unfavorable smells were a tree that clownfish are known to avoid, and eau de mom and dad.

Two of those probably need to be explained. You see, clownfish prefer to live near other clownfish, but they would rather avoid settling right next to mom and dad. This improves their chances of breeding successfully, while avoiding the genetically risky proposition of incest.

Once in the flume, the fish were allowed to pick the stream of water they favored. This process was repeated with the position of the streams swapped, and 20-40 fish were tested for each combination of rearing pH and smell. The results were dramatic.

The fish reared in normal ocean water behaved almost exactly as expected. They split 50:50 between neutral water sources, while they always preferred the favorable smelling water and always avoided the unfavorable smells, especially mom and dad. Meanwhile, the fish reared in pH 7.8 water seemed to prefer water with any smell, favorable or unfavorable. And the fish reared in pH 7.6 water? They didn't show a preference for any of the water types.

The researchers also verified that that the fish were not otherwise developmentally impaired, and that this was an effect of long term exposure to altered pH, not a chemical modification of the smell itself. Fish raised in pH 8.15 but tested in pH 7.6 behaved the same as fish raised in pH 8.15 and tested in pH 8.15. They found similar results with fish raised in pH 7.6 and tested in pH 8.15 as with fish raised in pH 7.8 and tested in pH 7.6. Finally, regardless of the pH they were raised in, all of the fish appeared normal in their ability to feed, swim, and search for a region to settle down in. There was no reported effect on their poop.

To assess what was causing this, the researchers stuck the fish into a scanning electron microscope to examine their nasal organs, but they were unable to find any significant physical changes. This suggests that the effect is due to an interference with the internal chemical signaling of the fish. Future acid tests may allow them to determine what is causing the problem, but, for the moment, they can't even tell if it is caused by the dissolved CO2 itself or the resulting change in pH.

That was a fascinating bit of reseach and sounds like a lot of work. They did not explore (yet) whether the gradual pH shift will be accomodated through selective breeding. In 100 years maybe the clownfish population will be adapted to the lower pH. Or not. TBD I guess.

Isn't that article's conclusion about lack of adaptability false? Just because the environment hasn't changed doesn't mean the genetic basis for adaptation is no longer there. I'm no geneticist but my reading leads me to believe that.

"To assess what was causing this, the researchers stuck the fish into a scanning electron microscope to examine their nasal organs, but they were unable to find any significant physical changes. This suggests that the effect is due to an interference with the internal chemical signaling of the fish"

This is directly contradicted by the fact that fish raised in the normal pH had no problem smelling when placed in the low pH tank.

Also, was this study blinded? There are huge opportunities for bias when you are trying to judge which "flume" a fish "prefers" to swim in. Given that the "developmentally impaired" fish functioned properly in all other measures, including activities that generally require a sense of smell - I'd look to a methodological explanation for their results.

Originally posted by joshv:"To assess what was causing this, the researchers stuck the fish into a scanning electron microscope to examine their nasal organs, but they were unable to find any significant physical changes. This suggests that the effect is due to an interference with the internal chemical signaling of the fish"

This is directly contradicted by the fact that fish raised in the normal pH had no problem smelling when placed in the low pH tank.

My reading was that they are still talking about developmental changes, not instant effects. That because they found no significant superficial physical differences, they propose that there may be differences in the internal components, specifically the chemical signalling mechanisms.

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Originally posted by joshv:Also, was this study blinded? There are huge opportunities for bias when you are trying to judge which "flume" a fish "prefers" to swim in. Given that the "developmentally impaired" fish functioned properly in all other measures, including activities that generally require a sense of smell - I'd look to a methodological explanation for their results.

Bias? Seriously? Because it's that hard to determine a preference of one side from the other? Perhaps the flume was divided so that the fish had to chose one side or the other and stick with it? Or are you just working under the assumption that scientists like noisy ambiguous data?

Though given your immediate jump to a methodological explanation I'll hazard a guess that that's your bias speaking. And that if you bother to reply you'll probably follow up with some standard pseudoskeptical talking points.

"Bias? Seriously? Because it's that hard to determine a preference of one side from the other? Perhaps the flume was divided so that the fish had to chose one side or the other and stick with it? Or are you just working under the assumption that scientists like noisy ambiguous data?"

Bias is bias, no matter how subtle. Perhaps the observer believes there is an effect. If they know which tank is the control and which is the experimental arm, it can influence their observations. This is scientific method 101 - nothing controversial here. If the experiment wasn't blinded in some form, that would be a serious point of criticism for the study. Perhaps they had some other methodological means of controlling for observational bias - I don't know. But I'd think some form of blinding would certainly be the best method.

My bias? I find the results contradictory, and find no mention of any sort of standard controls for observational bias. I explain that given that the only difference between their control and their experimental arms was in their "flume" experiments, and that in every other objective measure the fish were identical - it is perhaps reasonable to propose that there might be a methodological error in their "flume" experiment.

"And that if you bother to reply you'll probably follow up with some standard pseudoskeptical talking points."

The 'pseudoskeptical talking points' quip was, at the least, premature. And did not advance the discussion, my apologies.

Given that I don't think either of us is willing to put up the $10 to read the paper, we might have to settle for defining a few questions and seeing if anyone with institutional or other access will grace us with the answers.

To clarify: Is it your premise is that because the physical surface examination of the nasal organs did not find any differences, that you ascribe a higher probability to methodological error on the part of the researches than to a difference in action on the part of the fish?

"To clarify: Is it your premise is that because the physical surface examination of the nasal organs did not find any differences, that you ascribe a higher probability to methodological error on the part of the researches than to a difference in action on the part of the fish"

I would certainly look there for an explanation of the results. I have no idea how one would go about characterizing which flume of smell a fish prefers to occupy, but it sounds relatively difficult to me - and if the observation is not blinded, certainly highly open to influence by observational bias.

On the other hand, every other objective measure shows the fish to be identical - with the only explanation being "well, there must be some difference in the nasal apparatus that an electron microscope can't see". I'll certainly grant this is a possibility, though it seems unlikely. The simplest possible explanation is "you screwed up your experiment" - and it's the job of the research group (and good scientific journalists) to explain why this is not the case.

Originally posted by joshv:I have no idea how one would go about characterizing which flume of smell a fish prefers to occupy, but it sounds relatively difficult to me - and if the observation is not blinded, certainly highly open to influence by observational bias.

If you want to talk about strawmen, how about we talk about argument from incredulity?

Difficult, and highly open to bias? I propose:1) Bisect the upstream third of the flume with a non-permeable barrier.

2) Introduce the odorants into each side of the bisected flume, slightly downstream of the head inlet.

3) Release the fish at the bottom of the flume.

4) If the fish enters one side of the bisected flume to the exclusion of the other, mark in favour of that side. If over a suitable interval the fish enters both sides of the bisected flume, mark it as no preference.

I'm also going to go out on a limb and assume that someone who does fish research full time can think of something more robust than I have.

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Originally posted by joshv:On the other hand, every other objective measure shows the fish to be identical - with the only explanation being "well, there must be some difference in the nasal apparatus that an electron microscope can't see".

This is the second time you've characterized the SEM results as being "every other objective measure". The choice of such hyperbole to describe no "significant physical changes" in the nasal organ, takes a great degree of charity to read as anything other than deliberately deceptive. Particularly when they propose alternative explanatory mechanisms immediately afterward.

quote:

Originally posted by joshv:I'll certainly grant this is a possibility, though it seems unlikely. The simplest possible explanation is "you screwed up your experiment" - and it's the job of the research group (and good scientific journalists) to explain why this is not the case.

You are inferring that the shortest explanation is the simplest one. Is it your position that it is more likely that professional experimenters are ignorant of how to run a proper experiment. Than it is that the differences caused by developing in an moderately altered chemical environment may have effects that are not visible in an examination of the superficial physical structure?

"1) Bisect the upstream third of the flume with a non-permeable barrier.

2) Introduce the odorants into each side of the bisected flume, slightly downstream of the head inlet.

3) Release the fish at the bottom of the flume.

4) If the fish enters one side of the bisected flume to the exclusion of the other, mark in favour of that side. If over a suitable interval the fish enters both sides of the bisected flume, mark it as no preference."

Mmmm... Sciency. "upstream third", "slightly", "suitable interval".

Were the observations blinded - that's all I want to know. If they weren't, the science is suspect. Do you have some argument against blinding?

"This is the second time you've characterized the SEM results as being "every other objective measure". The choice of such hyperbole to describe no "significant physical changes" in the nasal organ, takes a great degree of charity to read as anything other than deliberately deceptive. Particularly when they propose alternative explanatory mechanisms immediately afterward."

Deliberate deception? The only observed differences were in the flume experiments. This is a fact. Do you dispute it?

"You are inferring that the shortest explanation is the simplest one. Is it your position that it is more likely that professional experimenters are ignorant of how to run a proper experiment. Than it is that the differences caused by developing in an moderately altered chemical environment may have effects that are not visible in an examination of the superficial physical structure?"

This is known as "Occam's razor". And yes, when your experimental results contradict every other observation, I'd question your ability to run an experiment.

If you would prefer to attack the choice of descriptive words, while completely ignoring their meaning, then I think we are done here. You claimed to want a discussion of the research presented, but then choose to spend your time avoiding actual discussion.

quote:

Originally posted by joshv:Were the observations blinded - that's all I want to know. If they weren't, the science is suspect. Do you have some argument against blinding?

Blinding is a tool for overcoming observational bias, it is not a box you must check off on the form that makes you 'sciency'. If the observed outcome is sufficiently clean and unambiguous, a lack of blinding would not invalidate the study. Would you question the results of a study on the relative buoyancy of balloons filled with helium versus balloons filled with water, when released in air, because the experimenter filled his own balloons and was thus not blinded?

Implying that because blinding is not 100% required for unambiguous results, and this I am 'against blinding', is sophistry. Would you like to have a discussion, or would you prefer to score points in a pointless game of debate theatre?

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Originally posted by joshv:Deliberate deception? The only observed differences were in the flume experiments. This is a fact. Do you dispute it?

I dispute your characterization of the experiment, your word choice is designed to deceive, entrap, and setup the 'gotcha' where you can turn to the audience with a look of bemusement.

In short, more sophistry and rhetorical tricks, if you have an argument to make, please do so.

The principal observed effect was in the flume experiment, and that was the core finding that the paper presented. I do not dispute that they claim to have observed a statistically significant difference in behavior.

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Originally posted by joshv:This is known as "Occam's razor".

You are incorrect, Occam's Razor states that, all else being equal, you should prefer the argument with the fewest assumptions. Your equating of 'fewest assumptions', with 'simplest', and further arguing that a shorter textual description is a simpler one, is flawed.

More than flawed however, it is a common trick of rhetoric used when trying to pull a con on a lay audience.

You are going to have to resort to making an actual argument at some point if you would like to convince anyone of anything.

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Originally posted by joshv:And yes, when your experimental results contradict every other observation, I'd question your ability to run an experiment.

If there was a large body of work in the field of fish raised in acidic environments and their olfactory navigation abilities, and if the findings in general were in disagreement with this, then you would be right to closely examine the experimental setup.

But then, you suggest attacking the experimenter instead of the experiment.

However this is the first study of this nature, and thus there is no 'every other observation' to contradict. There is only a single set of observations, made by a single team, and they are internally consistent.

You draw a false equivalence between the observed swimming results in the flume, and the negation of one possible cause of those results. Because of the negative SEM results, you say that the flume results 'contradict every other observation', the problem here is that they don't. They negate the hypothesis 'an acidic development environment produces gross physical changes to the nasal organ'. In and of itself this has little bearing on the core flume results, it merely negates a single possible causative mechanism.

If the SEM results were combined with additional results examining, for example: the chemical signalling pathways, the brain activity when exposed to different smells, or any of the myriad other explanatory mechanisms, then the flume result would be in contradiction.

Sheesh! If you read the original study you will see that they recorded which side of the flume the fish was on in 5 second intervals for a 2 minute period. Then they switched the sides the scented water was coming out of, waited 1 minute, then recorded another 2 minute period at 5 second intervals. They reported the percent of time the fish spent on each side of the flume. If the variation in these percentages were 55% (non-treated fish preferring flavor x) vs 51% (acid treated fish preferring flavor X) I might be skeptical. However, the results were never that ambiguous, for example: ~98% vs 1% (avoiding parent), 85% vs 1% (avoiding the unfavorable tree smell), 97% vs 90% (preferring anemone, I did say that the acid treated fish liked anything that smelled). 50% vs 75% (for the "neutral" grass smell) (These numbers are all approximate based on my reading them off a bar chart, the original numbers were not presented in the text.)

These are pretty clear results, and it would be hard for pure bias to report 98% vs 1%. In addition, all but one of the P values were <0.001. [and that one was <0.01).

Would it be better if it were double blind? Sure, is required to believe these results? I don't think so at all. In the case of many medical trials where the differences between the placebo and the test drug may be 60% vs 40% or less, and when there are reasons to believe that patient reporting may be influenced by subtle changes in a doctor's attitude, then double blind becomes a requirement. Recording which side of the flume a fish was swimming on? Not so much.

That said, it would have been nice to see a picture that showed the fish way over on one side of the flume. It is possible that if a fish was swimming right near the middle the results could be slightly influenced, I suspect that this was not the case or they would have made a point of describing how they distinguished which side the fish was on. The flume was divided by a physical barrier slightly upstream, so it is possible that they only recorded which side the fish was on if it swam up into one of these regions, but I doubt that was the case.